tbuckley/cabot-search huggingface dataset:
DIPG-28. IDENTIFICATION AND TARGETING OF METABOLIC VULNERABILITIES IN COMBINATION WITH PDGFRA-INHIBITION IN DIFFUSE MIDLINE GLIOMA
Authors: Sina Neyazi, Pruthvi S Gowda, Maria Trissal, Jay-Miguel Fonticella, Erika P Portero, John-Paul Haley-Read, Emily P Anderson, Nika N Danial, Mariella G Filbin
Journal: Neuro-Oncology (2023)

Abstract Diffuse midline gliomas (DMG) are driven by self-renewing, stem-like glioma cells stalled in an oligodendrocyte precursor cell (OPC)-like state that are characterized by high expression of PDGFRA. This lineage overexpression of PDGFRA, together with commonly found genomic amplifications and mutations in PDGFRA, has been shown to cooperate with H3-K27M mutations in tumor development. Therefore, targeting PDGFRA has long been proposed as a potential treatment strategy for DMG. Our previous work revealed that Avapritinib, a small molecule inhibitor of PDGFRA, is highly effective in DMG cells in vitro and in vivo. Following these preclinical findings, nine DMG patients were treated with Avapritinib for the first time. While 50% of all treated patients experienced a strong clinical response from Avapritinib, tumor progression and treatment escape were eventually observed in most patients. Here, we investigated which mechanisms DMG cells use to escape Avapritinib treatment and explored how these resistance mechanisms can be therapeutically exploited. Transcriptional analysis of a DMG cell line previously identified as Avapritinib-resistant revealed an upregulation of genes associated with fatty acid metabolism and oxidative phosphorylation (OXPHOS) following treatment with Avapritinib. Functional assays confirmed elevated OXPHOS in Avapritinib-resistant cells with significant (i) increase in mitochondrial energy transduction, (ii) increase in palmitate- (a product of fatty acid metabolism) driven oxygen consumption rate and (iii) greater incorporation of palmitate-derived carbons into the tricarboxylic acid cycle. These metabolic changes could be genetically reproduced in the same cell line after CRISPR-Cas9-mediated knockout of PDGFRA. To determine which therapies could target the dependency of Avapritinib-resistant cells on OXPHOS and fatty acid metabolism, we performed a metabolic drug screening and identified two metabolic drugs to have synergistic cytotoxic effects with Avapritinib. Together, we revealed distinct metabolic changes in Avapritinib-resistant DMG cells and showed that targeting these metabolic vulnerabilities might further increase the clinical benefit of Avapritinib.
EPEN-03. ZFTA-RELA LOCALIZATION DYNAMICS REGULATE TRANSCRIPTIONAL CONTROL
Authors: Amir Arabzade, Nic Laboe, Siri Ippagunta, Eric Emanus, Alisha Kardian, Stephen Mack
Journal: Neuro-Oncology (2023)

Abstract ZFTA gene fusions are the most frequent driver events in supratentorial ependymoma (EPN). ZFTA-fusion proteins act as oncogenic transcription factors (TFs) to aberrantly activate oncogenic transcriptional programs. Our data indicates that ZFTA fusion proteins form distinct punctate structures with heterogeneous patterns of liquid-phase-like characteristics. These condensates are closely associated with transcription as seen by nascent RNA FISH and recruitment of various members of the transcription machinery, such as BRD4, MED1, and RNA POLII. Our genomic footprinting data indicates that many key transcription factors, such as Sox9, share similar genomic occupancy patterns with ZFTA fusions suggesting that these transcription factors are potentially recruited to these condensates, establishing transcriptional hubs. Our mutagenesis studies reveal that the ZFTA zinc finger domain is indispensable for condensate formation, and may bridge ZFTA fusion proteins to DNA at unique genomic loci. Condensate-forming deficient mutants fail to establish the same transcriptional program and are unable to drive tumor formation. This mechanistic data has led to our efforts to establish drug-chemical screen approaches against ZFTA-RELA localization/dynamics to identify novel therapeutic targets.
Mapping interindividual dynamics of innate immune response at single-cell resolution
Authors: Natsuhiko Kumasaka, Raghd Rostom, Ni Huang, Krzysztof Polanski, Kerstin B. Meyer, Sharad Patel, Rachel Boyd, Celine Gomez, Sam N. Barnett, Nikolaos I. Panousis, Jeremy Schwartzentruber, Maya Ghoussaini, Paul A. Lyons, Fernando J. Calero-Nieto, Berthold Göttgens, Josephine L. Barnes, Kaylee B. Worlock, Masahiro Yoshida, Marko Z. Nikolić, Emily Stephenson, Gary Reynolds, Muzlifah Haniffa, John C. Marioni, Oliver Stegle, Tzachi Hagai, Sarah A. Teichmann
Journal: Nature Genetics (2023)

Common genetic variants across individuals modulate the cellular response to pathogens and are implicated in diverse immune pathologies, yet how they dynamically alter the response upon infection is not well understood. Here, we triggered antiviral responses in human fibroblasts from 68 healthy donors, and profiled tens of thousands of cells using single-cell RNA-sequencing. We developed GASPACHO (GAuSsian Processes for Association mapping leveraging Cell HeterOgeneity), a statistical approach designed to identify nonlinear dynamic genetic effects across transcriptional trajectories of cells. This approach identified 1,275 expression quantitative trait loci (local false discovery rate 10%) that manifested during the responses, many of which were colocalized with susceptibility loci identified by genome-wide association studies of infectious and autoimmune diseases, including the OAS1 splicing quantitative trait locus in a COVID-19 susceptibility locus. In summary, our analytical approach provides a unique framework for delineation of the genetic variants that shape a wide spectrum of transcriptional responses at single-cell resolution.
ATRT-13. ASSESSMENT OF MITOCHONDRIAL BIOENERGETICS AND FISSION-FUSION DYNAMICS IN ETMR
Authors: Evangelos Liapis, Annapurna Pamreddy, Allison Maas, Derek Hanson, Claire Carter
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND Embryonal tumor with multilayered rosettes (ETMR) is a deadly pediatric brain tumor with limited available therapeutic options. Dysregulated mitochondrial bioenergetics and dynamics have been linked to the initiation and progression of diverse human cancers, however, their role in ETMR pathobiology remains unknown. We used the primary ETMR cell line, BT183, and an in-house developed human neural stem cell (NSC) line to evaluate mitochondrial morphology, bioenergetics and fission-fusion dynamics involved in ETMR growth and aggression. METHODS Mitochondrial morphology was determined by widefield immunofluorescence and dSTORM super-resolution microscopy in BT183 and NSC monolayers and by TEM in BT183 and NSCs grown as neurospheres. Relative expression of mitochondrial proteins were quantified in BT183 and NSC lysates by western blotting. RESULTS Widefield immunofluorescence and super-resolution imaging revealed profound differences in mitochondrial morphology in BT183 compared to NSCs. BT183 mitochondria appear as fragmented, rounded structures, with little interconnectivity, while NSC mitochondria present a typical elongated, filamentous morphology. These findings were corroborated by TEM image analysis of BT183 and NSC neurospheres, which revealed additional morphological alterations in BT183 mitochondria at the ultrastructural level, including aberrant cristae structure. Western blot analysis of BT183 and NSC lysates showed up to 5-fold differential expression of mitochondrial OXPHOS, glycolytic and fission-fusion proteins in the two cell types. Fission promoters phospho-Drp1 and MFF displayed significantly higher expression in BT183 relative to NSCs, whereas fusion-inducing proteins Opa1 and Miro2 showed significantly lower differential expression in BT183. CONCLUSIONS BT183 cells exhibited striking mitochondrial fragmentation and differential expression of proteins involved in fission-fusion, OXPHOS and glycolysis when compared to normal human NSCs. Ongoing studies include the analysis of mitochondrial structure and morphology in patient samples to validate our in-vitro findings, in addition to in-vitro metabolic flux analysis and targeted protein depletion studies to identify druggable pathways based on mitochondrial function and dynamics.
Endoscopic treatment of complete stripping of esophageal mucosal and submucosal tissue layers
Authors: Francesca D’Errico, Maurizio Fazi, Jean-Loup Dumont, Gianfranco Donatelli
Journal: Endoscopy (2023)

A 45-year-old woman with advanced lower esophageal cancer was palliatively treated with placement of a partially covered self-expandable metal stent (PCSEMS) and chemoradiotherapy. Removal of the stent was needed 3 months later because of migration and stent ingrowth. We did not use argon plasma coagulation to burn the overgrowth tissue [1] at the edges of the stent because the stent had migrated and it was not possible to pass the scope through it ([Fig. 1]). Unfortunately, removal of the stent caused a stripping of the esophageal tissue layers. In detail, a 25-cm floppy "sleeve" of esophageal mucosa and submucosa was pulled out with the PCSEMS. The sleeve of tissue was intubated in order to visualize the de-epithelialized muscular layer ([Fig. 2]); this layer was not perforated as confirmed later by an upper barium swallow study ([Fig. 3]). The detached sleeve of esophageal tissue was pushed down again and a 12-cm fully covered SEMS and a nasoenteral feeding tube were inserted.
MDB-40. TRANSCRIPTOME-BASED DRUG REPURPOSING IN GROUP 3 MEDULLOBLASTOMA
Authors: David Doss, Ranjana Kanchan, Naveenkumar Perumal, Surinder Batra, Sidharth Mahapatra
Journal: Neuro-Oncology (2023)

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor. MB is divided into four molecular subgroups (SHH, WNT, Group 3 [G3], and Group 4 [G4]), each defined by unique genetic and molecular features. G3MB is the most aggressive subgroup, with the lowest five-year survival. Features conferring high risk to G3MB include frequent metastases at diagnosis, MYC amplification, and chromosome i17q. Outcomes for G3MB patients remain dismal, partly due to a lack of G3MB-specific therapies, defining an important gap in MB research. To address this gap, we employed a strategy of comparing the G3MB transcriptomic signature against a repository of chemical perturbagens (LINCS) to identify FDA-approved, blood-brain barrier penetrant drugs capable of reverting the disease signature to a more normal cerebellum-like state. Following preliminary in silico and in vitro screening, we identified four drugs - fluoxetine, sertraline, simvastatin, and nortriptyline - that passed screening criteria and demonstrated cytotoxicity against HDMB03 cells at low micromolar doses. Each compound also inhibited clonogenicity, impaired wound healing, and impeded cell migration and invasion. Further, each compound induced apoptosis as evidenced by annexin V and propidium iodide staining. Western blotting confirmed both dose- and time-dependent increases in the expression of key apoptotic markers. Since nortriptyline possessed the best cytotoxicity profile against HDMB03 cells, we interrogated the transcriptome of HDMB03 cells treated with nortriptyline to identify cancer-related pathways inhibited by drug treatment. Our data revealed strong inhibition of E2F transcription factors, which are critical to cancer cell cycle progression and proliferation. Notably, five of the six E2F transcription factors as well as a panel of E2F target genes (HN1, KIF4A, CDCA3, CDCA8, and SSRP1) are all specifically upregulated in G3MB. This study highlights the promise of drug discovery and repurposing using subgroup-specific transcriptomes, which may deliver effective treatments expeditiously to high-risk patients.
XRT-07. LATE-ONSET LYMPHOPENIA DURING CRANIOSPINAL RADIATION IS ASSOCIATED WITH AN INCREASED RISK OF TUMOR RECURRENCE IN NEWLY-DIAGNOSED PEDIATRIC MEDULLOBLASTOMA
Authors: Holly Lindsay, Michael Scheurer, Anthony Allam, Susan McGovern, Murali Chintagumpala, Arnold Paulino
Journal: Neuro-Oncology (2023)

Abstract A recent publication (PMID 33556478) demonstrated an increased risk of tumor recurrence in patients with newly-diagnosed pediatric medulloblastoma who have decreased lymphocyte counts early in their course of craniospinal radiation (CSI) (weeks 1 and 2). We set out to confirm this data in an independent population of pediatric patients with newly-diagnosed medulloblastoma. We analyzed 54 patients treated from 1997-2013. All patients that received radiation in or after 2007 received protons. Per standard of care, patients with clinically high-risk disease were treated with 36-39.6 Gy CSI while patients with standard-risk disease received 23.4 Gy. Total dose to the primary site of disease ranged from 54-55.8 Gy. The mean patient age at diagnosis was 8 years. 16/54 (30%) patients developed a recurrence at an average of 30.2 months after diagnosis. Lymphocyte counts were analyzed both as a continuous variable and as CTCAE v5.0 grades (grades 0-2 lymphopenia (ALC &amp;gt;/=500) compared to grade 3 and 4 (ALC&amp;lt;500/mm3)) using Cox regression. As expected, patients with M+ disease had increased risk of tumor recurrence (p=0.001). There was no correlation between risk of tumor recurrence and early lymphopenia (weeks 0-3 radiation). However, in both weeks 4 and 5 of radiation, decreases in ALC closely correlated with an increased risk of tumor recurrence (week 4 p=0.038, week 5 p=0.042). Similarly, patients with grade 3/4 lymphopenia at weeks 4 and 5 of radiation had an increased risk of tumor recurrence (week 4 p=0.040, week 5 p=0.008) compared to those with grade 0-2 lymphopenia. In summary, both M+ disease and lymphopenia during weeks 4 and 5 of radiation correlated with an increased risk of tumor recurrence in pediatric medulloblastoma.
HGG-14. RAS PATHWAY MEDIATED REGULATION OF HISTONE METHYLTRANSFERASE AND TUMOR SUPPRESSOR SETD2 IN PEDIATRIC HIGH GRADE GLIOMA
Authors: Angela Liou, Deborah Moran, Jacquelyn Dougherty, Kyra Harvey, Stephanie Brosius, Kay Labella, Thomas De Raedt
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND Pediatric High-Grade Gliomas (pHGG) are a significant cause of cancer-associated deaths in children. Hemispheric pHGG have frequent loss of the NF1 tumor suppressor (15%) causing aberrant RAS pathway activation. Intriguingly, loss of SETD2, a H3K36 methyltransferase, significantly co-occurs with loss of NF1. We found that nearly 80% SETD2-mutant pHGG have inactive NF1 (Pedcbio, 18/23 cases, 78%; odds-ratio 25; FDR adjusted P-value &amp;lt;0.001), suggesting a functional link between SETD2 and RAS signaling. NF1 is a GTPase-activating protein that negatively regulates the RAS oncogenic pathway. SETD2 is an epigenetic regulator and tumor suppressor that tri-methylates H3K36 on active gene bodies during transcription. We found that RAS pathway activation regulates SETD2 function. METHODS First, we used mass spectrometry to identify phosphorylation sites on SETD2 and generated phospho-mimetics/mutants to determine if these sites alter H3K36me3 levels. Second, we assessed if modulating downstream RAS effector pathways with MEK and PI3K inhibitors altered the phosphorylation of identified sites using the PhosTag gel electrophoresis assay. RESULTS We found that T2441 phosphorylation was upregulated upon MEK inhibition and that expressing T2441D phospho-mimetic and T2441A phospho-mutants had a differential effect on global H3K36me3 levels, implicating T2441 as an important regulatory node. Additionally, we identified that Cyclin Dependent Kinases are likely responsible for T2441 phosphorylation and are now investigating how RAS regulates these kinases. CONCLUSION To our knowledge it is the first time a functional connection between RAS pathway activation and SETD2 function has been found. It remains to be seen how of if RAS pathway activation alters the tumor suppressor function of SETD2 in pHGG.
Radiologic and endoscopic treatment for a disconnected pancreatic duct syndrome associated with percutaneous pancreatic fistula: achieving the “internalization of the fistula”
Authors: Ilaria Tarantino, Lucio Carrozza, Giacomo Emanuele Maria Rizzo, Salvatore Tammaro, Luigi Maruzzelli, Dario Ligresti, Mario Traina
Journal: Endoscopy (2023)

A 33-year-old man was referred to our institution for the management of a percutaneous pancreatic fistula after acute pancreatitis due to SARS-CoV-2 infection. He developed a peripancreatic collection (PPC), which was percutaneously drained because of infection. After resolution of the collection, percutaneous leakage from the main pancreatic duct (MPD) was observed. The patient underwent endoscopic retrograde cholangiopancreatography (ERCP) with biliary and pancreatic sphincterotomy, and placement of both pancreatic and biliary stents; however, the leak persisted.
IMG-08. VOLUMETRIC MEASUREMENT OF TUMOR SIZE OUTPERFORMS STANDARD TWO-DIMENSIONAL METHOD IN EARLY PREDICTION OF TUMOR PROGRESSION IN PEDIATRIC GLIOMA
Authors: Nastaran Khalili, Anahita Fathi Kazerooni, Sina Bagheri, Ariana Familiar, Karthik Viswanathan, Hannah Anderson, Debanjan Haldar, Jeffrey B Ware, Arastoo Vossough, Ali Nabavizadeh
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND Pediatric brain tumors (PBTs) are the leading cause of cancer-related death in children. Currently, based on the RAPNO guidelines, two-dimensional (2D) measurements are used to detect progression. However, due to the complex radiographic appearance of many PBTs, 2D measurements may not accurately reflect tumor growth. Here, we compared 3D, 2D, and qualitative radiologists’ interpretations in a group of PBTs to determine tumor progression. METHODS Six PBT patients (5 low-grade glioma, 1 ependymoma, age range at the time of baseline scan: 23-191 months) with an average of 5 imaging time points who had at least one episode of progression with subsequent surgery were included. Segmentation was performed on either T1 post-contrast or FLAIR, whichever that best identified the tumor. For 2D measurements, the two largest perpendicular diameters in a section that included the largest tumor component were measured. For the volumetric assessment, the tumors were manually segmented, and the volume was computed using the ITK-SNAP software. In multifocal tumors, only the tumor component that showed progression and underwent resection was included. A 25% and 41% increase in tumor size was considered as progression on 2D and 3D measurements, respectively. The time to progression (TTP) based on 2D vs 3D assessment as well as the official pediatric neuroradiologist interpretation based on the electronic health record report was compared. RESULTS In three of six patients, volumetric segmentation detected tumor progression at an earlier time point compared to 2D measurements and radiologists’ interpretation (median TTP: 134 compared to 503 for 2D and radiologist interpretation). For these three patients, the TTP based on 3D vs 2D assessment was: 252 vs 1299 days, 134 vs 503 days, and 80 vs 255 days. CONCLUSION Volumetric measurements can determine tumor progression earlier than the current standard method of 2D measurements and qualitative interpretation of radiologists in PBTs.
OTHR-07. CIRCULATING MIRNAS AS A NON-INVASIVE MONITORING TOOL FOR PEDIATRIC BRAIN TUMORS
Authors: Anne Coyle, Komal Rathi, Shehbeel Arif, Madison Hollawell, Christopher Beck, Namrata Choudhari, Tiffany Smith, Ammar Naqvi, Peter Madsen, Alexander Tucker, Phillip Storm, Adam Resnick, Jessica Foster, Mateusz Koptyra
Journal: Neuro-Oncology (2023)

Abstract Current management of pediatric brain tumor patients relies on imaging as the only non-invasive tool available for monitoring therapeutic response and tumor progression. Results are often inconclusive and unable to capture biological changes that presage progression on imaging. Circulating miRNA provides an attractive non-invasive platform because miRNA’s essential role in regulating gene expression, stability in bio-fluids, selective over-expression in tumors, and subsequent active release from tumors into the extracellular environment. We aimed to establish the prognostic and monitoring value of miRNA biomarkers for pediatric brain tumors. We performed miRNA profiling of CSF (n=33) and plasma (n=53) samples across a cohort of 54 pediatric brain tumors from 6 histologies (low grade glioma, ependymoma, germ cell tumor, medulloblastoma, atypical teratoid rhabdoid tumor and high-grade glioma). We utilized unsupervised consensus clustering, differential expression, and machine learning methods to explore miRNA biomarkers. Plasma miRNA signatures were correlated with WHO Grade while CSF signatures revealed a correlation with leptomeningeal disease and higher disease burden (p&amp;lt;0.05). The expression of circulating miRNA revealed histology-specific signatures for low grade glioma, medulloblastoma and germinoma in plasma and CSF (p&amp;lt;0.05). We identified miRNA signatures of significant translational value: (1) A medulloblastoma specific miRNA in CSF can serve as a potential biomarker for tumor monitoring (medulloblastoma specificity was confirmed in independent tissue-based dataset); (2) Pilocytic astrocytoma can be distinguished from high grade glioma in CSF; (3) Most intriguingly, in plasma, miRNA expression identified pilocytic astrocytoma that had relapsed in several months after initial intervention (p&amp;lt;0.05). These results support circulating miRNA in plasma and CSF as biomarkers of diverse clinical diagnostic and prognostic measures. Further utilization of this approach provides unique platforms to inform liquid biopsy-based management in the clinical setting.
MDB-17. THE PTBP2 SPLICING FACTOR IS ESSENTIAL IN GROUP 3/4 MEDULLOBLASTOMA
Authors: Veronika Caisova, Samuel Rivero-Hinojosa, Navya Chintala, I-An Lu, Zhang Huizhen, Aaron Mochizuki, Jianhua Yang, Brian Rood
Journal: Neuro-Oncology (2023)

Abstract Medulloblastoma (MB) is a common malignant pediatric brain tumor with significant heterogeneity among its four molecular subgroups WNT, SHH, Group 3 and 4. Group 3/4 MB are aggressive with a relatively poor prognosis indicating a critical need for novel therapeutic targets. Since the transcriptomic profiling of MB subgroups has not resulted in the expected identification of therapeutic potential, we focused on a proteomic approach. Building upon our previously published quantitative proteomic study, we selected 157 proteins significantly over-expressed in Group 3/4 MB. By using targeted loss-of-function CRISPR-Cas9 screen in four MB cell lines, we identified 17 essential genes shared by at least 2 of the cell lines. The PTBP2 splicing factor was essential to the survival of all cell lines. PTBP2 is an RNA binding protein involved in splicing regulation playing an important role during neuronal maturation. We hypothesize that persistent expression of PTBP2 results in a differentiation arrest and preservation of proliferative potential. To evaluate the role of PTBP2 in group 3/4 MB biology, we used single target CRISPR-Cas9 to stably knock-out PTBP2 expression in D556 and MB002 MB cells. We performed bulk RNA-seq and quantitative proteomics of these KO clones along with CLIP-seq to map PTBP2 targeted transcripts. We also performed phenotype assays in KO clones. RNA-seq in PTBP2 KO MB cells revealed 2,213 differentially expressed genes predominantly involved in neuronal differentiation and antigen presentation. These genes were cross-referenced with quantitative proteomics and eCLIP data. Phenotype assays of PTBP2 KO cells revealed reduced proliferation and migration compared to controls. To study the effect of PTBP2 on MB differentiation, we stimulated MB002 PTBP2 KO cells with FBS and observed increased surface attachment and differentiated morphology compared to controls. This suggests that persistent PTBP2 expression inhibits differentiation in MB cells with an attendant increase of proliferation activity.